International Journal of Oral Biology Original Article Int J Oral Biol 44:50-54, 2019 IJOB pISSN: 1226-7155 • eISSN: 2287-6618 https://doi.org/10.11620/IJOB.2019.44.2.50
Melatonin inhibits nicotinic acetylcholine receptor functions in bovine chromaffin cells Su-Hyun Jo1, Seung-Hyun Lee2, Kyong-Tai Kim3, and Se-Young Choi2*
1Department of Physiology, Institute of Bioscience and Biotechnology, BK21 Plus Graduate Program, Kangwon National University School of Medicine, Chuncheon 24341, Republic of Korea 2Department of Physiology, Dental Research Institute, Seoul National University School of Dentistry, Seoul 03080, Republic of Korea 3Department of Life Sciences, Division of Integrative Bioscience and Biotechnology, Pohang University of Science and Technology, Pohang 37673, Republic of Korea
Melatonin is a neurotransmitter that modulates various physiological phenomena including regulation and maintenance of the circadian rhythm. Nicotinic acetylcholine receptors (nAChRs) play an important role in oral functions including orofacial muscle contraction, salivary secretion, and tooth development. However, knowledge regarding physiological crosstalk between melatonin and nAChRs is limited. In the present study, the melatonin- mediated modulation of nAChR functions using bovine adrenal chromaffin cells, a representative model for the study of nAChRs, was investigated. Melatonin inhibited the nicotinic agonist dimethylphenylpiperazinium (DMPP) 2+ 2+ iodide-induced cytosolic free Ca concentration ([Ca ]i) increase and norepinephrine secretion in a concentration- 2+ dependent manner. The inhibitory effect of melatonin on the DMPP-induced [Ca ]i increase was observed when the melatonin treatment was performed simultaneously with DMPP. The results indicate that melatonin inhibits nAChR functions in both peripheral and central nervous systems.
Keywords: Melatonin, Nicotinic receptors, Calcium signaling, Neurotransmitter agents
Introduction Recently, crosstalk between melatonin and other signaling substances has been observed mainly in the central nervous Melatonin is a neurotransmitter that exhibits various physi- system [6,7]. ological functions ranging from circadian rhythm regulation Nicotinic acetylcholine receptors (nAChRs) are expressed to cancer cell metabolism [1,2]. Mainly synthesized from 5- in several oral tissues including submandibular ganglion cells hydroxytryptamine through serotonin N-acetyltransferase in [8], gingival epithelial cells [9], gingival keratinocytes [10], and the pineal gland, melatonin is secreted based on circadian gingival fibroblasts [11]. nAChRs have also been implicated rhythm and regulates whole body rhythmicity [3]. Melatonin in the development of maxillofacial skeletal muscle [12] and also participates in cell protection in conditions such as isch- ameloblasts [13]. Therefore, crosstalk between melatonin emia by blocking cell death and inhibiting autophagy [4,5]. The and nAChRs is a subject of interest. However, how melatonin cell-protective role of melatonin has been considered to occur modulates nAChR functions remains unclear. Thus, under- when high concentrations of melatonin act as antioxidants. standing the mechanism of nAChR inhibition by melatonin is
Received May 31, 2019; Revised June 9, 2019; Accepted June 11, 2019 *Correspondence to: Se-Young Choi, E-mail: [email protected] https://orcid.org/0000-0001-7534-5167 Copyright © The Korean Academy of Oral Biology CC This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by- nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Su-Hyun Jo, et al. Melatonin inhibits nicotinic functions important for understanding the neuroprotective role of mela- 3. Measurement of [3H]norepinephrine secretion tonin. In this study, the effects of melatonin on neurotransmitter [3H]norepinephrine secretion from chromaffin cells was mea- secretion by nAChRs were investigated in a peripheral nervous sured as previously described [18]. In brief, cells were loaded system model in which the nAChR acts as an excitatory neu- with [3H]norepinephrine (1 mCi/mL) in DMEM/F-12 for 1 hour rotransmitter. Subsequently, bovine adrenal chromaffin cells at 37℃ in a humidified atmosphere of 95% room air and 5% with a nAChR-mediated neurotransmitter secretion system CO2. The cells were incubated in Locke’s solution (154 mmol/L were used. Chromaffin cells act as postganglionic cells that NaCl, 5.6 mmol/L KCl, 10 mmol/L glucose, 2.2 mmol/L CaCl2, secrete norepinephrine by receiving preganglionic cholinergic 1.2 mmol/L MgCl2, and 5 mmol/L HEPES buffer adjusted to inputs from sympathetic systems [14]. Thus, the neurotrans- pH 7.4) to measure the basal secretion. The cells were stimu- mitter secretion modulated by nAChRs has been intensively lated with DMPP for 15 minutes and the medium was used studied using chromaffin cells [15,16]. In the present study, to analyze the secreted [3H]norepinephrine. Residual/total [3H] crosstalk between melatonin and nAChRs was investigated to norepinephrine was extracted by adding 10% trichloroacetic understand the peripheral neuromodulating effects of melato- acid. The radioactivity was measured using a scintillation coun- nin. ter. The amount of [3H]norepinephrine secreted was calculated as the percentage of total [3H]norepinephrine content. Materials and Methods 4. Free Ca2+ concentration measurement 1. Materials 2+ 2+ The [Ca ]i was determined using the fluorescent Ca in- Melatonin, dimethylphenylpiperazinium (DMPP), and sulfin- dicator Fura-2/AM, as previously reported [19]. Briefly, the pyrazone were obtained from Sigma-Aldrich (St. Louis, MO, bovine chromaffin cell suspension was incubated in serum- USA). Fura-2 pentaacetoxymethyl ester (Fura-2/AM) was free DMEM/F-12 media with 4 mM Fura-2/AM for 60 minutes purchased from Molecular Probes (Eugene, OR, USA). [3H] at 37℃ under continuous stirring. Sulfinpyrazone (250 mM) was Norepinephrine was purchased from PerkinElmer NEN (Bos- added to all solutions to prevent Fura-2 leakage. Fluorescence ton, MA, USA). DMEM/F-12 and penicillin/streptomycin were ratios were monitored with dual excitation at 340 and 380 nm purchased from GIBCO (Grand Island, NY, USA). Bovine calf and emission at 500 nm. Calibration of the fluorescent signal in 2+ serum and horse serum were obtained from HyClone (Logan, terms of [Ca ]i was performed [20] using the following equa- UT, USA). tion: 2+ [Ca ]i = KD[(R – Rmin)/(Rmax – R)](Sf2/Sb2) 2. Chromaffin cell preparation where R is the ratio of fluorescence emitted by excitation at
340 and 380 nm. Sf2 and Sb2 are the proportionality coefficients Chromaffin cells were isolated from bovine adrenal medulla at 380 nm excitation of Ca2+-free Fura-2 and Ca2+-saturated using the two-step collagenase digestion as previously de- Fura-2, respectively. Rmin, the minimal fluorescence ratio, was scribed [17,18]. For the [3H]norepinephrine secretion assay, measured at a condition of 4 mmol/L EGTA, 30 mmol/L Trizma isolated bovine chromaffin cells were plated in 6-well plates at base, and 0.1% Triton X-100. Then Rmax, the maximal fluores- a density of 5 × 105 cells per well. For the cytosolic free Ca2+ cence ratio, was measured at a condition of 10 mmol/L Ca2+. 2+ concentration ([Ca ]i) measurement, cells were transferred to 100-mm culture dishes (1 × 107 cells per dish). The cells were 5. Data analysis maintained in DMEM/F-12 containing 10% bovine calf serum and 1% penicillin/streptomycin. The cells were incubated in a All quantitative data are expressed as means ± standard humidified atmosphere of 95% room air and 5% CO2. The cul- error of the mean. Data were analyzed using Origin software ture medium was changed every three days. (Origin Lab, Northampton, MA, USA). The results were ana- lyzed using the unpaired Student’s t-test and p ＜ 0.05 was considered statistically significant.
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Results Discussion
To examine the regulation of nAChR functions in melatonin, In this study, the effect of melatonin on the regulation of first, the effects of melatonin on nAChR-induced norepineph- nAChR functions was investigated to understand crosstalk rine secretion in bovine adrenal chromaffin cells were exam- between melatonin and nAChRs. Results showed preincuba- ined. The nAChR-specific agonist, DMPP, successfully induced tion with melatonin inhibited DMPP-mediated norepinephrine 2+ the preloaded norepinephrine secretion. Preincubation with secretion, and melatonin inhibited DMPP-mediated [Ca ]i in- melatonin inhibited DMPP-mediated norepinephrine secretion in a concentration-dependent manner (Fig. 1). 2+ The catecholamine secretion is triggered by elevated [Ca ]i 400 2+
(nM) level. The effects of melatonin on DMPP-induced [Ca ]i increase i ] 350
2+ 2+ was investigated. DMPP evoked an increase in [Ca ]i in Fura-
[Ca 300 2-loaded bovine adrenal chromaffin cells. Under this condi- 2+ 250 tion, melatonin inhibited DMPP-induced [Ca ]i increase in a concentration-dependent manner (Fig. 2). In addition, the 200 2+ inhibitory effect of DMPP-induced [Ca ]i increase was more 150 pronounced with the melatonin analogue, 2-iodomelatonin. DMPP-induced in 100 Vehicle Next, whether melatonin acts directly on nAChRs or by ac- Melatonin 50 2-iodomelatonin tivating melatonin-specific receptors was examined. The time
Increase 0 course of melatonin-mediated inhibition of DMPP-induced 6 5 4 3 2 2+ [Ca ]i increase was investigated. Melatonin simultaneously Log [Preincubated agonist] (M) 2+ suppressed the inhibition of the DMPP-induced [Ca ]i increase 2+ Fig. 2. Melatonin inhibits DMPP-evoked [Ca ]i increase in bovine adrenal when preincubated with melatonin for 30 seconds (Fig. 3). The chromaffin cells. Fura-2-loaded cells were treated with 20 μM DMPP in the result indicates melatonin does not require preincubation to presence of the indicated concentrations of melatonin, 2-iodomelatonin, or 2+ exert an inhibitory effect and may directly inhibit nAChRs. vehicle. Peak levels of DMPP-induced Ca influx were quantitatively ana- 2+ lyzed and depicted as % of the DMPP-induced [Ca ]i increase. Each point is the mean ± standard error of the mean (n = 3–5). 2+ 2+ DMPP, dimethylphenylpiperazinium; [Ca ]i, free Ca concentration.
i
]
14 2+ 100
[Ca 12 80
10 60
control)
control)
of
of
norepinephrine
8 DMPP-induced (% 40
(%
of 6 20 Vehicle
secretion Melatonin
4 Vehicle Inhibition
DMPP-induced Melatonin 0 0 2 4 6 8 10 12 2 6 5 4 3 Incubation time (min) Log [Melatonin] (M) Fig. 3. Time course of melatonin-mediated inhibition of DMPP-evoked 3 2+ Fig. 1. The effects of melatonin on [ H]norepinephrine secretion in bovine [Ca ]i increase. Fura-2-loaded cells were treated with 20 μM DMPP after adrenal chromaffin cells. [3H]Norepinephrine-loaded chromaffin cells were preincubation with 3 μM melatonin, or vehicle, for the indicated time. Peak treated with 20 mM dimethylphenylpiperazinium (DMPP) in the presence levels of DMPP-induced Ca2+ influx were quantitatively analyzed and de- 3 2+ of the indicated concentrations of melatonin or vehicle. The secreted [ H] picted as % of the DMPP-induced [Ca ]i increase. Each point is the mean norepinephrine is expressed as % of total [3H]norepinephrine. Each point ± standard error of the mean (n = 3–6). 2+ 2+ is the mean ± standard error of the mean (n = 3). DMPP, dimethylphenylpiperazinium; [Ca ]i, free Ca concentration.
52 www.kijob.or.kr Su-Hyun Jo, et al. Melatonin inhibits nicotinic functions crease in bovine adrenal chromaffin cells. nAChRs are expressed in submandibular ganglion cells and Melatonin controls cell functions in various ways. Crosstalk regulate salivary secretion [8]. In addition, nAChRs are ex- between melatonin and nAChRs can be divided into the fol- pressed in maxillofacial skeletal myocytes to regulate tensile lowing two types: (1) Melatonin directly inhibits nAChRs. Mela- stress [12]. In particular, the nAChRs present in ameloblasts tonin has direct binding affinity to nAChRs in the noradrenergic are involved in enamel formation by differential expression nerve terminal of rat vas deferens [21] and guinea pig submu- during the tooth morphogenesis stage [13]. Therefore, the ef- cous plexus [22]. In addition, melatonin was shown to exert a fect of melatonin on oral functions is very important in dental neuroprotective effect by inhibiting alpha-7 nAChR in organo- science. Recently, neuromodulation of nAChRs in the central typical hippocampal cultures [23]. (2) Melatonin modulates via nervous system has received much clinical attention. Choline melatonin-specific G protein-coupled receptors. In general, esterase inhibitors, which block the degradation of acetyl- similar to amino acid derivative neurotransmitters, melatonin choline and increase its concentration, have been shown to activates melatonin-specific G protein-coupled receptors, MT1 improve cognitive function in Alzheimer’s disease and mild and MT2 [24]. In cerebellar granule cells, picomolar melatonin cognitive impairment; nAChRs play an important role in this inhibited nAChR-mediated cation current through MT1 and mechanism [6,27]. Therefore, the results from this study will MT2 receptors [25]. However, the detailed mechanism of how not only contribute to a better understanding of the physiology melatonin regulates other components, including the peripher- of melatonin and nAChRs, but also reaffirm the possibility for al nervous system, remains unclear. Therefore, identifying mo- various clinical applications of melatonin as a nAChR modula- lecular targets that receive the neuroactive effects of melatonin tor. is important to better understand the effects of complex and diverse functions of melatonin. In the present study, the dose Acknowledgements curve and time course of the inhibitory effects of melatonin 2+ on melatonin-induced [Ca ]i increase was investigated, and This work was supported by the National Research Foundation results showed inhibition was in a relatively high concentration of Korea (2018R1A5A2024418 to S.Y.C.; 2015R1D1A1A01057549 range within a very short time period (i.e. within several sec- to S.H.J.) and by 2017 Research Grant from Kangwon National onds). These results indicate the melatonin-mediated nAChR University (520170430). inhibition in chromaffin cells may be caused by a direct inhibi- tion independent of G protein-coupled receptors. This direct Conflicts of Interest action was observed in other mechanistic effects of melatonin, and we recently showed the voltage-sensitive calcium chan- No potential conflict of interest relevant to this article was nels inhibitory effect of melatonin acts in a melatonin-specific reported. receptor-independent manner [26].
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